This application incorporates by reference the material in the ASCII text file named “Seq_List_LCTI_200040USP3_ST25.txt”, which was created on Feb. 10, 2016, and has a file size of 8,781 bytes.
The present disclosure relates to methods for producing certain desired dopaminergic cells from human pluripotent stem cells (hESCs) and for predicting their phenotypic maturation after transplantation to the brain based on molecular markers. These dopaminergic cells can then be used in stem cell based therapies such as for treating neurodegenerative diseases including Parkinson's disease. Also disclosed are kits for practicing the methods. Very generally, the stem cells are cultured on a substrate of laminin-111 (or other recombinantly produced laminins) and exposed to a variety of cell culture mediums to produce the dopaminergic cells with much higher efficiency than previously attained.
Laminins are a family of heterotrimeric glycoproteins that reside primarily in the basal lamina. They function via binding interactions with neighboring cell receptors on the one side and by binding to other laminin molecules or other matrix proteins such as collagens, nidogens or proteoglycans. The laminin molecules are also important signaling molecules that can strongly influence cellular behavior and function. Laminins are important in both maintaining cell/tissue phenotype, as well as in promoting cell growth and differentiation in tissue repair and development.
Laminins are large, multi-domain proteins, with a common structural organization. A laminin protein molecule comprises one α-chain subunit, one β-chain subunit, and one γ-chain subunit, all joined together in a trimer through a coiled-coil domain. The twelve known laminin subunit chains can form at least 15 trimeric laminin types in native tissues. Within the trimeric laminin structures are identifiable domains that possess binding activity towards other laminin and basal lamina molecules, and membrane-bound receptors. FIG. 1 shows the three laminin chain subunits separately. For example, domains VI, IVb, and IVa form globular structures, and domains V, IIIb, and IIIa (which contain cysteine-rich EGF-like elements) form rod-like structures. Domains I and II of the three chains participate in the formation of a triple-stranded coiled-coil structure (the long arm).
There exist five different alpha chains, three beta chains and three gamma chains that in human tissues have been found in at least fifteen different combinations. These molecules are termed laminin-1 to laminin-15 based on their historical discovery, but an alternative nomenclature describes the isoforms based on their chain composition, e.g. laminin-111 (laminin-1) that contains alpha-1, beta-1 and gamma-1 chains. Four structurally defined family groups of laminins have been identified. The first group of five identified laminin molecules all share the β1 and γ1 chains, and vary by their α-chain composition (α1 to α5 chain). The second group of five identified laminin molecules, including laminin-521, all share the β2 and γ1 chain, and again vary by their α-chain composition. The third group of identified laminin molecules has one identified member, laminin-332, with a chain composition of α3β3γ2. The fourth group of identified laminin molecules has one identified member, laminin-213, with the newly identified γ3 chain (α2β1γ3).
Human embryonic stem (hES) cells hold promise for the development of regenerative medicine for a variety of diseases, such as spinal cord and cardiac injuries, type I diabetes and neurodegenerative disorders like Parkinson's Disease. A stem cell is an undifferentiated cell from which specialized cells are subsequently derived. Embryonic stem cells possess extensive self-renewal capacity and pluripotency with the potential to differentiate into cells of all three germ layers. They are useful for therapeutic purposes and may provide unlimited sources of cells for tissue replacement therapies, drug screening, functional genomics and proteomics.
Stem cell based therapies and treatments for neurodegenerative diseases are expected to reach clinical trials soon. However, a major hurdle remains in generating standardized good manufacturing practices (GMP)-grade human pluripotent stem (hPS) cell-based progenitors that upon transplantation will mature and function in the adult brain. As cells are transplanted as immature progenitors and full maturation into functionally integrated neurons requires 6 months or longer in vivo, transplanted cells cannot be assessed for functionality prior to transplantation, and there is a lack of markers that reliably predict yield and maturation of the immature progenitors.
As a surrogate for markers predicting functional properties, the field has relied upon assessing transplanted progenitors by expression of genes and proteins developmentally linked to the generation of certain neuronal subtypes. Since it is uncertain if these markers are predictive of terminal differentiation and functional maturation, the progenitors must therefore be taken through lengthy in vivo functional assessment in order to control for batch-to-batch variation and therapeutic efficacy. This can pose a significant hurdle for the generation of cells for larger cohorts of patients and for launching a standardized stem cell product to the clinic.
To reduce the in vivo studies needed to be performed during the cell developmental process, it will thus be necessary to identify a validated set of markers that can predict the in vivo performance of the stem cell product prior to transplantation. Being able to predict an in vivo graft outcome at the progenitor stage may also lead to the production of standardized cell products from variable input sources, such as from patient-derived cells or hPS cell banks with HLA-haplotyped donors.
Parkinson's disease is a particularly interesting target for stem cell based therapies due to the relatively focal degeneration of a specific type of mesencephalic dopamine (mesDA) neuron. Proof-of-concept that cell replacement therapy for Parkinson's disease can be successful has been obtained in a number of clinical trials using fetal cells.
Recent developments have resulted in a better understanding of the developmental and cellular ontogeny of mesDA neurons from the floor plate of the ventral mesencephalon, and this knowledge has materialized into research grade differentiation protocols that result in the formation of ventral mesencephalon progenitors from hPS cells. In contrast to older protocols, these dopaminergic neurons of ventral mesencephalon origin have been shown to survive, mature, and acquire appropriate functional properties in animal models of Parkinson's disease. Moreover, grafts generated from these protocols do not result in cell overgrowths or tumor formation, thus making them suitable candidate cells for stem cell replacement therapies.
In current ventral mesencephalon differentiation protocols, mesencephalic floor plate markers LMX1A, FOXA2, OTX2, and CORIN are commonly used to confirm the mesDA identity of progenitors in vitro prior to grafting, but a new study has revealed that these and several other commonly used ventral mesencephalon markers are also co-expressed in diencephalic progenitors of the subthalamic nucleus. Thus, they appear to be suboptimal markers in protocols for the generation of enriched mesDA neurons, as they are clearly not specific to the mesDA domain.
For the purposes of regenerative medicine and for modeling human neural cells, there is a desire to develop methods that allow derivation and culturing of pluripotent stem cells under chemically defined, xeno-free, pathogen-free, and for reproducible differentiation of these cells into neural cells with regenerative capacity. Desirably, such methods should provide large quantities of such differentiated cells. Further, it would be desirable to develop methods for predicting successful graft outcomes of transplanted progenitors in an animal model.